I'm recalculating the RPKM value of a RNASeq data on Rsubread through featureCounts function, and I'd like to know if should I use just the "assigned" reads or the total reads, including "unassigned ambiguity, multimapping..." (see below), in the RPKM formula. Looking for the answer in forums and in the Mortazaviet al.(2008), I've just find out that "N is the total number ofmappable reads in the experiment". So, could anybody please help in this regards?
RPKM = N/(L*T)
N: number of reads assigned to a gene
L: length of the gene (kb)
T: total mapped reads (Millions)
T_reesei_F24.1_GGCTAC_L008_R1_001.cleanreads.fastq.gz_tophat2.F24h.1_accepted_hits.bam Assigned 32270962 Unassigned_Ambiguity 6896 Unassigned_MultiMapping 116803 Unassigned_NoFeatures 10751746 Unassigned_Unmapped 0 Unassigned_MappingQuality 0 Unassigned_FragementLength 0 Unassigned_Chimera 0
Thanks in advance!